The regulated secretion of hormones and transmitters by endocrine and nerve cells occurs by similar processes | involving the Ca2+-dependent fusion of secretory vesicles with the plasma membrane. The importance of | regulated exocytosis for nervous and endocrine system function has motivated efforts to understand the underlying molecular mechanisms. Recent work identified several proteins including syntaxin, VAMP and SNAP25 (termed SNAREs) as playing fundamental roles. However, it is unclear what these roles are and j whether SNAREs function in the docking, priming, triggering or fusion stages of exocytosis. We have been abl[unreadable] ! to reconstitute stages of SNARE-dependent regulated exocytosis in a membrane preparation of PC 12 cells that I consists of docked secretory vesicles on plasma membranes. Exocytosis of docked vesicles proceeds through ATP-dependent primingand Ca2+-dependent fusion steps. The ATP-dependent priming step involves the action j of NSF (N-ethylmaleimide-sensitive factor) and the synthesis of phosphatidylinositol(4,5)bisphosphate (PIP2). j Using this membrane preparation, which is enriched for proteins involved in regulated exocytosis, we will establish biochemical correlates of function by determining the role of SNARE proteins at post-docking stages o exocytosis, and by identifying mechanisms that regulate SNARE protein function at these stages. Our aims will be to: 1. Clarify mechanisms that regulate PIP2 levels, and determine the role of PIP2 in regulating SNARE protein function; 2. Establish the nature of SNARE protein complexes at the priming, triggering and fusion stages of exocytosis, and determine the roles of NSF and Muncl8/n-Secl in regulating SNARE complex assembly and disassembly; 3. Determine the role of a novel Ca2+-dependent synaptotagmin-SNAP25 interaction in Ca2+-triggered fusion; and 4. Extend the scope of mechanistic studies to the poorly-understood process of I vesicle docking. This work will provide new insights on the precise role of essential proteins in specific stages o: ! regulated exocytosis, and will enhance our understandingof the molecular basis for Ca2+-dependent transmitter I and hormone secretion. __ _ _ i